Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin

Singh, Utkarsha A.; Kumari, Mukta; Iyengar, Soumya

doi:10.1186/s12575-018-0077-6

Cited by 68 publications

(56 citation statements)

References 22 publications

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“…The pooled samples of aspirate were thawed on ice, gently agitated and 5 mL aspirated for DNA isolation using a modification of a previously described technique . Briefly, the sample was centrifuged for 30 min at 10,000 g and 4°C.…”

Section: Methodsmentioning

confidence: 99%

Culture‐independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease

Beste

Lawhon

Chamoun‐Emanuelli

et al. 2019

Equine Veterinary Journal

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Summary Background Horses with bacterial sinusitis frequently undergo empirical treatment with antimicrobials, however, in some cases bacterial culture of the affected sinus is used to direct therapy. Data regarding which organisms are part of the commensal microbiota of the equine sinus are lacking making it difficult to interpret culture results and guide empiric antimicrobial selection. Objectives Our objectives were to describe the bacterial and fungal microbiota of the paranasal sinuses in clinically normal horses using culture‐dependent and independent approaches and to compare the bacterial culture and susceptibility patterns of normal horses with those from horses affected with primary and secondary sinusitis. Study design Experimental study and descriptive retrospective review of case records. Methods Sinus washes were collected from 23 healthy horses. Washes were submitted for routine culture and susceptibility testing and DNA was isolated for next generation sequencing of bacterial and fungal marker genes. For clinical cases of sinusitis, medical records from 2010 to 2017 were reviewed and horses diagnosed with primary and/or secondary sinusitis were included. Results The paranasal sinus cavity hosts multiple bacterial and fungal organisms. The bacterial microbiota of healthy horses consists largely of uncultivable, aerobic bacteria. While few anaerobes were isolated from normal horses, the majority of clinical cases resulted in growth of anaerobic organisms with no difference in the proportion of aerobic and anaerobic bacteria isolated from clinical cases. Main limitations Small sample size in both populations of horses and heterogeneity of the population prevent a more in‐depth analysis. Conclusions The microbiota of the paranasal sinuses of horses consists primarily of aerobic bacteria and fungal organisms, the majority of which are uncultivable via common clinical methods. Anaerobic bacteria are found in the majority of horses with clinical sinusitis. These findings suggest anaerobic bacteria are associated with sinusitis and their presence should be considered when treating horses with sinusitis.

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Section: Methodsmentioning

confidence: 99%

Culture‐independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease

Beste

Lawhon

Chamoun‐Emanuelli

et al. 2019

Equine Veterinary Journal

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“…Extractions using chelating beads provided a cost-effective alternative to more expensive salting-out approaches, such as Qiagen DNeasy kits. Chelating extractions, however, can also produce lower quality DNA and may include suspended impurities (Singh et al, 2018). Campbell et al (2015) did show that GT-seq can be conducted using DNA from chelating extractions but did not directly compare results using multiple extraction protocols.…”

Section: Discussionmentioning

confidence: 99%

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

Bootsma

Gruenthal

McKinney

et al. 2020

Preprint

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Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus) and discuss trade-offs associated with different development and genotyping approaches. Our GT-seq panel was developed using restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We then conducted simulations to test the utility of loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We conducted three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. Optimization focused on reducing variation in amplification rate among loci and minimizing the proportion of off-target sequence, both of which are important considerations for developing large GT-seq panels. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was likely unnecessary, and (3) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings should aid in their construction in myriad taxa.

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“…Combining multiple panels could facilitate genotyping of > 1,000 loci rather than a few hundred, providing greatly increased power for kinship analysis and GSI (Baetscher et al, 2018;. Chelating extractions, however, can also produce lower quality DNA and may include suspended impurities (Singh, Kumari, & Iyengar, 2018). Campbell et al (2015) did show that GT-seq can be conducted using DNA from chelating extractions but did not directly compare results using multiple extraction protocols.…”

Section: Further Optimization Of the Gt-seq Protocolmentioning

confidence: 99%

A GT‐seq panel for walleye (Sander vitreus) provides important insights for efficient development and implementation of amplicon panels in non‐model organisms

Bootsma

Gruenthal

McKinney

et al. 2020

Molecular Ecology Resources

View full text Add to dashboard Cite

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals. Development of GT-seq panels is nontrivial, but studies describing trade-offs associated with different steps of GT-seq panel development are rare. Here, we construct a dual-purpose GT-seq panel for walleye (Sander vitreus), discuss trade-offs associated with different development and genotyping approaches, and provide suggestions for researchers constructing their own GT-seq panels. Our GT-seq panel was developed using an ascertainment set consisting of restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We conducted simulations to test the utility of all loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We then performed three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (i) inexpensive Chelex extractions performed well for genotyping; (ii) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was probably unnecessary; and (iii) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings and suggestions should aid in their construction in myriad taxa.

show abstract

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin

Cited by 68 publications

References 22 publications

Culture‐independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease

Culture‐independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

A GT‐seq panel for walleye (Sander vitreus) provides important insights for efficient development and implementation of amplicon panels in non‐model organisms

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